One of the missing high-impact opportunities in immuno-oncology has been the inability to maximally exploit the synergy between conventional chemotherapy and novel immunotherapy. The premise of the current proposal is that successful chemo-immunotherapy critically depends on eliciting robustly immunogenic, pro-inflammatory antigen-presenting cells that can cross-present tumor antigens. In tumors at baseline, however, the cross- presenting dendritic cell (DC) population is profoundly defective, and the default immune response is tolerance and immunosuppression. How to change this defect has been unclear. This proposal tests the hypothesis that Bruton?s Tyrosine Kinase (BTK) in tumor-associate myeloid cells represents a powerful and previously unrecognized innate-immune system checkpoint, that dominantly suppresses the emergence of a key population of highly pro-inflammatory monocyte-lineage DCs during chemotherapy. If the suppression by this myeloid BTK checkpoint can be overcome, then dying tumor cells now become able to spontaneously elicit large numbers of inflammatory monocytic DCs, which drive robust and sustained anti-tumor immunity. Aim 1 will use genetically- defined models to identify the specific myeloid-lineage cells in tumors that are suppressed by BTK. It will test the hypothesis that when the BTK checkpoint is blocked during chemotherapy, these immature myeloid cells now become able to rapidly mature into inflammatory DCs. These activated DCs reprogram the ?cold? tumor milieu into a ?hot? pro-inflammatory microenvironment, and re-activate anergic CD8+ effector T cells. Aim 2 will test the hypothesis that the BTK checkpoint is normally dominant in tumors, because it is constantly driven by key tolerogenic signals such as tumor-induced Tregs and local TGF?. However, if this repressive BTK pathway is blocked, then immature myeloid cells in tumors become able to mature into inflammatory APCs in response to signals from dying tumor cells. Aim 3 will test the hypothesis that the tolerogenic indoleamine 2,3- dioxygenase (IDO) pathway is tightly coupled to the BTK checkpoint in myeloid cells. During chemotherapy, IDO acts as a direct cell-intrinsic metabolic signaling pathway downstream of BTK, allowing BTK to suppress the maturation-inducing mTOR pathway in DCs. IDO is also elicited by dying tumor cells during chemotherapy as a paracrine pathway in plasmacytoid DCs and tumor-associated macrophages, which activate Tregs and produce local TGF? to reinforces the BTK checkpoint. Thus, blocking both BTK and IDO together creates potent mechanistic synergy, and allows robust anti-tumor immune activation during even modest chemotherapy. Relevance: If the proposed strategies are successful, this will bring a novel, high-impact approach to chemo-immunotherapy, focused on eliciting a crucial population of immunogenic antigen-presenting cells. BTK- inhibitor drugs are approved, and IDO-inhibitor drugs are in clinical trials, so translation to the clinic would be rapid and direct.